REACTION SEQUENCE: The sequence of steps in the biosynthesis of cholesterol from lanosterol is being investigated by two experimental approaches. In the first approach, the multi-component enzymic system contained in liver microsomes is incubated under conditions that lead to the accumulation of biosynthetic intermediates. In the second approach, enzymes that catalyze these reactions are isolated from microsomal preparations, purified, and characterized. With a combination of these two approaches, the sequence of one-half of the individual reactions in the twenty-step formation of cholesterol from lanosterol has been elucidated. Under study during the -11 year have been the delta 8 yields delta 7-isomerase, the NADPH-dependent 3-ketosteroid reductase, and the 32-demethylase, all of which are now at various stages of purification. Interruption of metabolism of the 14 alpha-methyl group of lanosterol has been achieved. REGULATION: During the past year, we have continued the study of the regulation of rates of the twenty terminal reactions of cholesterol biosynthesis. The oxidative attack of the 4 alpha-methyl group has been found to be rate-determining. A noncatalytic protein has been isolated from the postmicrosomal supernatant fraction and purified to homogeneity. This cytosolic protein stimulates methyl sterol oxidase and, in the presence of the protein and product, inhibition of methyl sterol oxidase can be produced in vitro by the addition of cholesterol and cholesterol esters to the incubations. The cytosolic protein also stimulates HMG-CoA reductase, and end product inhibition of reductase has been produced in broken cell preparations. MICROSOMAL ELECTRON TRANSPORT: Parallel studies on the effects of cytosolic proteins on the mixed function oxidation of stearyl-CoA to oleyl-CoA, as well as methyl sterol oxidase, have revealed stimulation by two different soluble proteins. Control of each oxidase appears to be separate.